Soft Ionization Based on Conditioned Glow Discharge for Quantitative Analysis

ABSTRACT

One aspect of the disclosure provides a method of mass spectrometric analysis that includes producing either glow discharge within a noble gas between 3-100 mBar pressure, sampling and conditioning glow discharge products within a gas flow through a conductive channel, removing charged particles while transferring excited Ridberg atoms, and mixing conditioned discharge products with analyte flow within an enclosed chamber at elevated temperatures above 150° Celsius for producing a Penning reaction between analyte molecules and Ridberg atoms. The method further includes sampling, by a gas flow, said analyte ions for mass spectrometric analysis, and at least one of the following steps: (i) removing charge within said conditioning channel; (ii) coaxially mixing of analyte flow with the flow of conditioned plasma; and (iii) cooling of the mixed flow within a sonic or supersonic jet for reducing the region of Penning ionization to cold jet.

TECHNICAL FIELD

This disclosure relates to mass spectrometry. More specifically, thisdisclosure relates to mass spectrometry using a soft and quantitativelyionizing source based on conditioned glow discharge ionization.

BACKGROUND

Development of soft ionization methods, like Electrospray (ESI) andMatrix Assisted Laser Desorption/Ionization (MALDI) have extended thefield of mass spectrometric analysis to wide class of labilecompounds—such as peptides, nucleotides, proteins, and lipids—and havetriggered the development of wide range of biological and medicalapplications. The methods are known to be limited to compounds whichreadily produce ions in liquid, such as ionic salts, and to polarcompounds, readily producing protonated MH⁺ and deprotonated ions(M−H)⁻. The range of soft ionized compounds was extended to semi-polarcompounds with introduction of Atmospheric Pressure Chemical Ionization(APCI) and Atmospheric Pressure Photo Ionization methods (APPI) methods.One shortcoming, however, is that these soft ionizing methods are notfully quantitative because the nature of the analyzed compounds defineand vary both the ionization efficiency and the gas-phase stabilityagainst competitive ion molecular reactions.

On the other pole, the truly quantitative method of electron impactionization (EI), wherein compound vapors are ionized by electronbombardment, has long existed. The ionization efficiency stays constantin wide range of analyte concentration, usually measured as sample loadthrough a gas chromatograph (GC). Typically, linearity is sustained froma limit of detection (LOD) being as low as ten femtograms (10 fg) inmost sensitive instruments and up to ten nanograms (10 ng) load range(i.e. at least within six orders of dynamic range). The ionizationefficiency (i.e. response versus load) is mostly non-dependent onmolecule nature, and stays independent on other coeluting compounds andmatrix. This allows the EI method to be uniform across chemical classesand truly quantitative. The EI method, however, is limited tosemi-volatile compounds, and it is not soft is not soft because itproduces extensive fragmentation.

In addition to coupling with an EI method source, GC separation has beencoupled to alternative and notably softer ionization methods—such aschemical ionization (CI) and field ionization (FI)—which provide moreintensive molecular peak. The CI technique, however, is also prone tomatrix and mutual interference effects. With this ionization method,both ionization efficiency and spectra content strongly depend oninstrumental parameters. Thus, the CI technique is not considered to befully soft, truly quantitative, or capable of providing library spectra.And the CI technique is also considered “dirty” due to the rapidcontamination of the ion source. The FI method is frequently regarded asa soft ionization method; however, it is tricky, unstable, andinsensitive with typical detection limit of only around one hundredpicograms (100 pg). For this reason, the FI method has not been widelyadopted.

Photo ionization (PI) and photo-chemical ionization (APPI) methods aremuch softer compared to EI, though still produces fragments for highlyfragile compounds. Schlag describes in U.S. Pat. No. 4,570,066, which isfully incorporated herein by reference, that multi-photon ionization forlaser desorbed nucleotides and short peptides, along with their coolingby a supersonic jet with subsequent multi-photon resonance ionization,which appeared to be moderately soft. The method was not widely adopteddue to selective ionization, insufficient softness, and a limited classof analyzed compounds.

Glow discharge has been long employed in mass spectrometry for elementaland organic analysis, such as in F. W. Aston, MASS SPECTRA AND ISOTOPES,2^(nd) edition, Longman Green, N.Y., 1942, which is fully incorporatedherein by reference. In Hunt et. al, Anal. Chem vol. 47 (1975) 1730(which is fully incorporated herein by reference), a Taundsen glowdischarge was proposed for ionizing dopant gas in a CI source. U.S. Pat.No. 4,321,467 (which is fully incorporated herein by reference) proposesorganics ionization in flow-afterglow at mbar-level gas pressures. VGAnalytics introduced liquid samples via Thermospray interface andinduced a glow discharge in the fore-vacuum region, as described in U.S.Pat. No. 4,647,772 and U.S. Pat. No. 4,794,252 (each of which is fullyincorporated herein by reference). In U.S. Pat. No. 4,849,628 (which isfully incorporated herein by reference), McLuckey suggested sampling ofliquid vapors from atmospheric pressure region into a glow dischargewithin a fore-vacuum stage at one to ten mbar gas pressure. Lubman etal. suggested ionized gaseous and liquid samples within Helium glowdischarge at atmospheric pressure, as described in Applied Spectroscopy,44 (1990) 1391 and Anal. Chem. 64 (1992) 1426 (each of which is fullyincorporated herein by reference). Numerous groups have attempted toimprove the softness and analytical merits of the glow dischargeionization sources. In spite of large variety of glow discharge sources,the employed ionization methods are split between two categories: (a)direct ionization and (b) chemical ionization.

Direct ionization in glow discharges occurs primarily due to Penningionization by excited metastable of noble gases, while minor channelscorrespond to charge transfer from discharge ions and to electron impactionization. Such ionization is likely to be quantitative, but harsh. Asan example, Bertand et.al in JASMS, 5 (1994) 305 (which is fullyincorporated herein by reference), exposed organic analytes to mBar glowdischarge and demonstrated linear signal response within five orders ofdynamic range, while obtaining spectra with softness varying from EI toCI spectra. Both sensitivity and softness appear strongly dependent onthe analyzed compound and on the parameters of ion source. Adding dopantgases improves the intensity of molecular protonated ions and formsspectra similar to CI ones, as shown by Mason et al. in Int. J. MassSpectrom. Ion Proc. 91 (1989) 209 (which is fully incorporated herein byreference).

The chemical ionization in glow discharges (or by sampled products ofglow discharge) occurs primarily due to proton transfer from protonatedwater clusters, originating from ubiquitous water traces in technicalpurity gases. Proton transfer from water clusters has been intentionallypromoted in a controlled proton transfer reaction (PTR) massspectrometry as described by Hansel et al. in Int. J. Mass Spectrom. IonProc. 149 (1995) 609 (which is fully incorporated herein by reference).Lubman et al. ionized gaseous and liquid samples within Helium glowdischarge at atmospheric pressure, as described in Applied Spectroscopy,44 (1990) 1391 and Anal. Chem. 64 (1992) 1426 (each of which is fullyincorporated herein by reference). Adding water with liquid samplessubstantially improved softness of organic spectra. However, the protontransfer reactions caused non-linear signal per concentration responseand non-uniform ionization. Efficiency of ionization varied within threeorders of magnitude between analyzed compounds. Proton affinity is knownto depend on compound polarity, which explains the non-uniformionization between chemical classes. Operation at atmospheric (ascompared to mbar) pressures increases the role of ion molecularreactions, which explains mutual analyte interferences and matrixsuppression effects, even at large excess of the charging agent.

A DART glow discharge method has been described in Andrade et.al, Anal.Chem, 80 (2008) 2646-2653 (which is fully incorporated herein byreference), where volatile compounds are mixed with glow discharge inhelium at atmospheric pressure. Though the method describes Penningionization as the main mechanism, large number of gas collisions lead tosignificant distortions by ion molecular reactions. Thus, this alsoproduces protonated ions for polar compounds and, hence, is also proneto discrimination and interference effects. Thus, glow dischargeionization is likely to be either (a) quantitative but harsh at directGD ionization, or (b) soft but not quantitative at chemical ionization,primary implemented by proton transfer from water clusters.

In WO 2012/024570 (which is fully incorporated herein by reference), theinventors of this disclosure attempted to soften direct ionization inglow discharge by using a conditioner (i.e. a conductive tube forcontrolling plasma residence time prior to sampling discharge productsinto an ion-molecular reactor with analyte). GC inlet, purified gases,and clean materials were used to reduce the amount of quenchingparasitic vapors. However, the conditioning appears strongly dependenton trace amount of vapors, strongly reduces efficiency of ionization, sothe choice remained the same—either quantitative or soft.

SUMMARY

This disclosure provides the long waited combination of soft andquantitative ionization accomplished by a conditioned glow dischargemethod. The method also inducing effective identification using a U.S.National Institute of Standards and Technology (NIST) library search.This method accomplishes such a desirable ionization by: (a) generatinglarge fluxes of metastable noble gas atoms at mBar glow discharge; (b)sampling those metastable particles and suppressing charged particles;and (c) using gaseous cooling for analyte molecules simultaneous withPenning ionization in cold supersonic gas jet at limited number of gascollisions.

One astonishing aspect of the method provided by this disclosure is theeffect of the gaseous cooling on the analyte ion stabilization. Coolingof analyte and of surrounding Argon gas dramatically reduces the amountof fragments and provides spectra with molecular M+ ions only. In animplementation, the supersonic jet cooling is arranged at coaxialsampling of analyte molecules and of the glow discharge products.

Gaseous cooling to overweighs the excitation effects at glow dischargeformation of metastable atoms, and soft ionization occurs at a widerange of discharge parameters. This allows reaching large fluxes ofmetastable atoms, while using very moderate conditioning for removal ofcharged particles. As a result, the efficiency of ionization approachesunity (ratio of formed ions per injected analyte molecule) and appearsuniform between chemical classes of analytes.

Efficient ionization by the method is reached within a supersonic jetwith small number of neutral collisions, which is estimated to about100. Thus, even at reasonable technical purity of gases and materials,the source strongly reduces the amount of cluster formation and of otherparasitic ion molecular reactions with analyte ions.

The novel method presented by this disclosure is a conditioned glowdischarge ionization method that called Cold GD. Experimental tests ofthis novel Cold GD method have confirmed:

1.) Soft ionization for labile molecules—such as alkanes, phthalates,nitroses, and fatty acids—that are known to form intensive fragmentationin methods like EI, CI, Cold EI, and PI;

2.) Formation of molecular M+ ions only, which simplifies spectraidentification, unlike prior art glow discharge and PI sources whichform both M+ and MH+ ions;

3.) Uniform response between wide range of classes, confirmed foralkanes, PAH, PCB, phthalates, and nitro-containing compounds;

4.) Linear response within at least four orders of dynamic range,between one picogram (1 pg) and ten nanogram (10 ng) load via GC column;

5.) Absence of chemical discrimination, interference, and matrix effectsat chemical matrix fluxes under 10 ng/sec;

6.) Ability to form NIST type of fragments at ion excitation within theion transfer interface, which is characteristic for M+ ions and isuseful for compound identification based on library spectra; and

7.) Fast response of the ion source, matching the speed of GC×GCanalysis.

The combination of softness, true quantification, large dynamic range,and fast response opens numerous opportunities for improved analyticalmethods in the field of petroleomics and metabolimcs, so as utilizingmultidimensional separations, like GC×GC-MS, GC−IMS−MS, GC−MS−MS forcomplex mixtures analysis. Some implementations and their novel aspectsfor quantitative analysis of complex mixtures and detection ofultratraces are described in this disclosure.

One aspect of the disclosure provides a method of mass spectrometricanalysis including producing either RF or DC glow discharge within anoble gas at gas pressure between 3 and 100 mBar, sampling andconditioning glow discharge products within a gas flow through aconductive channel for removing charged particles while transferringexcited Ridberg atoms, mixing conditioned discharge products withanalyte flow within an enclosed chamber at elevated temperatures above150° Celsius for producing a Penning reaction between the analytemolecules and the Ridberg atoms to generate ions of the analyte, andsampling the analyte ions (by a gas flow) for mass spectrometricanalysis. The method also includes at least one of the following steps:(1) removing charge within the conditioning channel caused by chargingof an insulating surface protruding through the conditioning channel;(2) coaxially mixing of analyte flow with the flow of conditionedplasma; and (3) cooling of the mixed flow within a sonic or supersonicjet for reducing the region of Penning ionization to cold jet.

Implementations of the disclosure may include one or more of thefollowing optional features. In some implementations, the molecular ionsof analyte are partially fragmented either at an ionization event or ata step of controllable collisional induced dissociation, and obtainedfragment spectra are compared with library spectra of electron impactfor analyte identification and for a structure elucidation. In someexamples, the method further includes introducing less volatilecompounds within a liquid or solid matrix to extend the range of analytevolatility whereat a matrix flux remains under 10 ng/sec and where theanalyte sample is brought to a gas phase bone one of the followingsteps: (a) applying a rapid thermo desorption; (b) applying pulsed laserdesorption; (c) applying nebulization a liquid sample with removal ofsolvent vapors by side or counter gas while passing through aerosol; and(d) applying nebulization at a flow rate under 10 nL/min pas capillaryelectrophoresis or nano liquid chromatography. The method may furtherinclude a step of upstream tandem chromatographic separation of the listcomprising: (i) GC−GC; (ii) LC−GC; (iii) LC−LC; (iv) LC−CE.

In some implementations, for the purpose of enhanced selectivity atanalysis of complex mixtures, the method further includes a step of massor ion mobility selection of parent ions prior to the step of ionfragmentation. The method may further include a step of varyingfragmentation energy. In some examples, for the purpose of enhancedanalysis selectivity, the method further includes a step of adding areagent gas into the region of conditioned glow discharge, thusconverting Ridberg ions into reagent ions. In some implementations, themethod further includes a step of mass-spectrometric analysis of theionized analyte ions with a high resolution multi-reflectingtime-of-flight mass spectrometer, operating in the regime of frequentencoded pulsing.

Another aspect of the disclosure provides a method of mass spectrometricanalysis including quantative and soft ionizing in a conditioned glowdischarge ion source, alternated in time measuring of molecular mass andfragmentation of molecular ions, and compound identifying by comparingwith library of electron impact spectra. This aspect of the disclosuremay include one or more of the following optional features. In someexamples, the method further includes at least one step of the group:(i) an upfront multi-stage chromatographic separation of analytemolecules; (ii) an upfront mass separation of molecular ions; (iii) ionmobility separation of molecular ions.

Yet another aspect of the disclosure provides a method for assembling anion source for a mass spectrometry apparatus that includes providing areactor chamber, arranging a glow discharge chamber adjacent to thereactor chamber, providing a tubular electrode extruding into aninterior of the glow discharge chamber, arranging a sampling nozzle atan outlet end of the reactor chamber, providing a capillary for sampleintroduction, and arranging a mechanical fluid pump in a location toallow evacuation of gas. The mechanical fluid pump evacuates the gasfrom the glow discharge chamber past the sampling nozzle. The reactorchamber defines a sampling conditioning channel. The capillary passesthrough the glow discharge chamber, protrudes through the tubularelectrode, and at least partially passes through the reactor chamber.

This aspect of the disclosure may include one or more of the followingoptional features. In some implementations, the method further includescharging an insulated surface that protrudes through the samplingconditioning channel. In some examples, the gas evacuated by themechanical fluid pump is a noble gas, and the noble gas is pressurizedwithin the glow discharge chamber prior to evacuation.

A fourth aspect of the disclosure provides an ion source for a massspectrometry apparatus that includes a reactor chamber that defines asampling conditioning channel, a glow discharge chamber residingadjacent to the reactor chamber, a tubular electrode that receives avoltage and extrudes into an interior of the glow discharge chamber, asample nozzle residing at an outlet end of the reactor chamber, acapillary for sample introduction, and a mechanical fluid pump. Thecapillary passes through the glow discharge chamber, protrudes throughthe tubular electrode, and at least partially passes through the reactorchamber. The mechanical fluid pump resides in a location to allowevacuation of gas, and the mechanical fluid pump evacuates the gas fromthe glow discharge chamber past the sampling nozzle.

This aspect of the disclosure may include one or more of the followingoptional features. In some examples, the ion source further includes aninsulated surface that protrudes through the sampling conditioningchannel. In some implementations, the gas evacuated by the mechanicalfluid pump is a noble gas, and the noble gas is pressurized within theglow discharge chamber prior to evacuation.

Yet another aspect of the disclosure provides an analytical method thatincludes quantitatively soft ionizing an analyte in a conditioned glowdischarge ion source, alternating-in-time measuring of molecular massand fragmentation of molecular ions, and identifying compounds bycomparing with a library of electron impact spectra.

This aspect of the disclosure may include one or more of the followingoptional features. In some implementations, the ionizing step includesproducing a glow discharge from a noble gas at an elevated pressurebetween 3-100 mBar, conditioning the glow discharge through a samplingconditioning channel, and ionizing an analyte by mixing the analyte withthe conditioned glow discharge at an elevated temperature. In someexamples, an insulated surface within the sampling conditioning channelis charged to remove charge of a flow through the sampling conditioningchannel. The step of ionizing an analyte may include a coaxial mixing ofa flow of the analyte with a flow of the glow discharge. In someexamples, a step of cooling the ionized analyte within a jet occursafter the step of ionizing an analyte. In some implementations, the stepof ionizing an analyte occurs at the throttle of a sampling nozzle.

Yet another aspect of the disclosure provides a method of conditionedglow discharge ionization including arranging an enclosed ionizationchamber for minimal outgassing, heating the ionization chamber to anelevated temperature above 250 degrees Celsius, feeding a noble gas intothe ionization chamber at a flow rate between one hundred millilitersper minute and one thousand milliliters per minute, evacuating the gasthrough a sampling conditioning channel, inducing glow discharge betweena tubular electrode and a counter-electrode, inserting an insulatingsurface through the tubular electrode to stabilize the glow dischargeinducement, creating a gas flow through the sampling conditioningchannel to move the induced glow discharge products, protruding theinsulating surface through the sampling conditioning channel,introducing an analyte sample, mixing the analyte sample with the glowdischarge products in close vicinity or within a throttle of a samplingaperture, providing local cooling of the analyte sample within a gascooling jet, wherein the gas cooling jet is formed within the samplingaperture, and directing products to a mass spectrometer.

This aspect of the disclosure includes may include the followingoptional feature. The insulating surface may be a quartz capillary, andthe quartz capillary may introduce the analyzed sample.

DESCRIPTION OF DRAWINGS

FIGS. 1-3 are schematic views of example chemical glow dischargeionization apparatuses.

FIG. 4 is a schematic view of an example conditioned glow dischargeionization apparatus.

FIGS. 5A-5B are schematic views of example apparatuses for applying aConditioned Gas Discharge (CGD) method for mass spectrometric analysisof complex mixtures of semi-volatile compounds.

FIGS. 6A-6B show plots relating to effects of a jet used for cooling theCGD method accomplished by the apparatuses of FIGS. 5A-5B.

FIG. 7 shows examples of spectra for representative compounds createdutilizing the CGD method accomplished by the apparatuses of FIGS. 5A-5B.

FIG. 8 shows an array of spectra for hexachlorobenzene at variousenergies of collisional induced dissociation obtained with the CGDmethod accomplished by the apparatuses of FIGS. 5A-5B.

FIG. 9 shows a plot of for survival (relative intensity of molecular M+ions to the total spectrum intensity) of various types of analyte versusion injection energy into an RF ion guide incorporated into theapparatuses of FIGS. 5A-5B.

FIGS. 10A-10D show results of a fragment spectra interpretation.

FIG. 11 shows spectra related to analysis of fatty acids methyl esters(FAME).

FIGS. 12A-12C show comparisons between a total ion current for CGD andEI methods for a MegaMix sample.

FIG. 13 shows a plot of bi-logarithmic signals versus HCB load onto agas chromatograph (GC) column illustrating capabilities of CGD forquantitative analysis.

FIG. 14 shows a table comparing characteristic of various ionizationmethods to characteristics of the CGD method accomplished by theapparatuses of FIGS. 5A-5B.

FIG. 15 is a flow chart detailing exemplary operations for making theapparatuses of FIGS. 5A-5B.

FIG. 16 is a flow chart detailing exemplary operations for performingmass spectrometric analysis including the CGD method accomplished by theapparatuses of FIGS. 5A-5B.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

This disclosure describes methods and apparatuses for mass spectrometryutilizing a soft and quantitatively ionizing source based upon glowdischarge ionization. Referring first to FIGS. 5A-5B, a conditioned glowdischarge ionization mass spectrometry apparatus 50 and an accompanyingconditioned glow discharge (“CGD”) method, which are described in moredetail hereinafter, are disclosed. Turning to FIG. 14, a review of themerits of various ionization methods is presented. The CGD method(summarized in row 1411)—which may be accomplished, for example, byutilization of apparatus 50—is compared with other ionization methods(summarized in rows 1401-1410). FIG. 14 indicates that some methods doprovide ionization with particular characteristics (indicated by ‘Yes’).other methods do not provide ionization with such characteristics(indicated by ‘No’), and some method provide a limited ability of acertain characteristic (indicated by ‘Some’).

While soft ionizing methods such as ESI and MALDI (along with APCI andAPPI), refer to rows 1401-1403, have extended the range of compoundsthat may be analyzed with mass spectrometry, these soft ionizing methodscannot be considered fully quantitative since the nature of the analyzedcompounds define and vary both the ionization efficiency and the gasphase stability against competitive ion molecular reactions. A fullquantitative analysis could be possible by accounting for variableefficiency of ionization; however, the ionization efficiency appearsdependent on ion source parameters, on the analyzed solution, on mutualanalyte interference, and on matrix effects. Signal changes nonlinearwith analyte concentration and depends on concentration of coelutingcompounds and chemical matrix. In the attempt of obtaining quantitativedata, one should introduce an internal standard (preferably isotopiclabeled) for each class of analyte and, preferably, for each analyzedcompound. Accounting for the wide range of known labile compounds, whichexceeds 50 million records in the Chemical Abstract Database, theinternal standard approach appears unpractical for routine use of thesesoft ionization methods, except for particularly important analysescases. Thus, widely used soft ionizing methods—ESI, MALDI, APCI andAPPI—cannot be considered truly quantitative, as documented in FIG. 14.

Contrary to widely represented opinion, this disclosure claims thatthose soft ionization methods do not provide library spectraidentification based on fragment spectra produced in tandem massspectrometers (see right column in FIG. 14). Although peptides could beidentified based on a library search, the spectra are not reproduciblebetween instruments with strong variations of relative fragmentintensities. Although fragment spectra for other compound classes couldbe interpreted, confident assignment between functional isomersbelonging to different chemical classes would only be accomplished inrare circumstances. This looks to be the fundamental property ofprotonated ions.

Electron impact ionization (EI), refer to row 1404, is anotherionization method. As illustrated in FIG. 14, this method is trulyquantitative, but it is not soft; it produces extensive fragmentation.While it is not a soft method, the fragmentation produced by the EImethod has been utilized as a benefit of the overall analytical method.Electron energy is kept at 70 eV in order to obtain standard fragmentspectra, which is highly reproducible between various instruments. Gaschromatography (GC) is employed to separate analytes in time, so thatindividual fragment spectra could be deconvoluted (i.e. extracted basedon fragments' simultaneous appearance in time). Extracted EI spectra arethen submitted for comparison with a library of standard EI spectra forcompound identification (see right column of FIG. 14). The method allowsidentification of several hundreds of compounds per single gaschromatograph-mass spectrometer (GC−MS) run. Two-dimensional gaschromatography (GC×GC) extends the limit to thousands of analyzedcompounds per single run. However, the utilization of a GC−MS systemwith an EI ionization source chokes when sample complexity exceeds tensof thousands and individual fragment spectra, which can no longer beseparated. Harshness of the EI method also limits the analysis to labileanalyte molecules. For a wide range of particularly fragile (thoughsemi-volatile) analytes, like alkanes, phthalates, nitroses, and manyother classes, the EI spectra do not provide sufficient molecular peakintensity which affects the identification. In this case, an entiremolecule of an analyte can be confused with its own subset. To this end,GC−MS applications have been longing for a complementary soft ionizationtechnique.

The chemical ionization (CI) method, refer to row 1405, is not trulyquantitative, and despite being softer than the EI method has onlylimited abilities to provide a soft ionization; it is not a truly softmethod. And the CI method does not provide for library spectra. Thefield ionization (FI) method, refer to row 1406, also has only a limitedability to provide soft ionization. And it has not been widely adopteddue to its instability and unfavorable detection limit.

The softness of the EI method has been improved with analyte coolingwith a gas jet, as described by Amirav in U.S. Pat. No. 5,055,677, whichis fully incorporated herein by reference. However, this improved EImethod, refer to row 1407, does not provide truly soft ionization, butrather provides a reduction of fragment intensity and moderateenhancement of molecular ion intensity. And another shortcoming of thiscold EI method is that the degree of fragmentation varies withexperimental parameters.

The photo ionization (PI) method and related methods (such as APPI),refer to row 1408, are softer than the EI method, but still producefragments when ionizing fragile compounds. Thus, while possessing alimited ability to produce soft ionization, it has a limited associatedrange and is not truly quantitative. With the use of sealed UV lampsthese methods becomes suitable for wide range of moderately polarcompounds. The PI method has been utilized for detection after GC, asdescribed in U.S. Pat. No. 4,377,749, U.S. Pat. No. 4,413,185, and U.S.Pat. No. 4,398,152, each of which is fully incorporated herein byreference. In SU1159412 (fully incorporated herein by reference) andmultiple scientific papers Revelsky et al. suggested using the PI methodat atmospheric conditions for GC−MS analysis. Photo ionization isaccompanied by damping of internal energy at atmospheric pressure whichmakes it much softer compared to vacuum UV ionization. To enhanceefficiency of ionization there are added dopant vapors of acetone orbenzene; thus promoting the formation of molecular M⁺ and protonated MH⁺ions with little amount of fragmentation and with detection limitbetween one to ten picograms (1-10 pg). Some variations of the methodare suggested in U.S. Pat. No. 5,541,519 and U.S. Pat. No. 5,338,931,each of which is fully incorporated herein by reference. However, themethod may cause confusion in spectra interpretation with formation ofeither M⁺ or MH⁺ ions. Present experimentation shows that the methodforms ionic clusters and moderate amount of fragment ions. Besides, theAPPI method fails at analysis of saturated hydrocarbons (SHC) and doesnot ionize compounds with high ionization potential (PI) above 9-11 eV,like small mass halogenated compounds. The ionization efficiency dependson competition for proton with dopant, and there is a much higher spreadof compound dependent ionization efficiency in the PI method compared tothe EI method. Ionization efficiency varies with dopant concentrationand is prone to chemical interference and suppression mechanisms. Thus,PI cannot be considered as a truly quantitative method. Same is evenmore related to LC-APPI sources, where competition for charge isinevitable at presence of large amount of solvents.

Glow discharge ionization, refer to rows 1409-1410, are generally splitinto direct glow discharge ionization (refer to row 1409) and chemicalglow discharge ionization (refer to row 1410). Direct glow dischargeionization methods may be fully quantitative, but fail to achieve a softionization. Chemical glow discharge ionization methods may achieve softionization, but fail to by fully quantitative. WO 2012/024570, which isfully incorporated herein by reference, presents some advances, butultimately fails to achieve both quantitative and soft ionization.

As a summary of FIG. 14, soft ionization methods, based on protontransfer, like ESI, MALDI, APCI and APPI, are widely adopted forproviding molecular mass information. However, they are not uniform inionization, not truly quantitative, are only applicable to a limitedclass of analyte compounds, and do not form library fragment spectra. Incontrast, the electron impact (EI) ionization method is trulyquantitative, provides uniform ionization, and forms library fragmentspectra. However, it is not soft, which presents problems foridentifying labile molecules and limits the sample complexity, sincerich spectra have to be deconvoluted in GC−MS analysis. Complementary toEI methods—such as CI, cold EI, and FI—improve softness but presenttheir own practical problems. Photoionization method with sealed UV lampand dopant assisted chemical ionization are significantly softer, butare not uniform in ionization efficiency and are prone to interferenceeffects, typical for methods with protonated molecular ions. Glowdischarge methods can be as robust as the EI method; however, in pastimplementations there was always a compromise between softness andquantification features. One can chose between softness andquantification, but could not achieve both. Hence, the CGD method of row1411 of FIG. 14 represents a novel ionization method with advantageouseffects not presented in any other single ionization method of rows1401-1410.

Attempts have been made to improve upon the ionization methods of rows1401-1410, such as by utilizing a gas dampening or a gas jet coolingprocess. A gas dampening of these ionization methods may have directeffects on the softening of the ionization. For example, SU1159412(which is fully incorporated herein by reference) proposes to use anatmospheric pressure dampening for softening of the photo ionizationmethod. U.S. Pat. No. 6,504,150 (which is fully incorporated herein byreference) proposes gaseous dampening of MALDI generated ions. Lubmanet. al has demonstrate much softer GD spectra at atmospheric pressure inApplied Spectroscopy, 44 (1990) 1391 and Anal. Chem. 64 (1992) 1426(each of which is fully incorporated herein by reference). However, theoperation at elevated gas pressures inevitably promotes chemicalionization at presence of technical clean gases (typical impuritiesabove 1E-6) and source materials, which in turn causes formation ofprotonated ions by a non-quantitative ionization, resulting innon-uniform response between chemical classes.

Also, expanding upon the above-discussed gas jet cooling (see also row1407), earlier publications suggest that prior collisional cooling ofanalyte molecules in supersonic jets have a positive effect on ionstability at subsequent “harsh” ionization at much lower gas pressures.U.S. Pat. No. 4,570,066 explores this technique for multi-photonionization, and U.S. Pat. No. 5,055,677 explores this technique for EIionization. Both of these references are fully incorporated herein byreference. Despite such a positive effect in some of the other methods,jet cooling appears insufficient at direct glow discharge ionization inglow discharge, as described and explained by McLuckey et.al, Anal.Chem, 60 (1988) 2220 (which is fully incorporated herein by reference).Cooling by gas jet means stabilizes molecules at charge transfer;however, this has not mitigated ion fragmentation during direct glowdischarge ionization methods. Thus, utilization of either gas dampeningor jet cooling has not resulted in soft and quantitative glow dischargeionization. As such attempts have failed to remedy the shortcomingillustrated in rows 1409-1410, ionization methods that combine softnesswith quantification are needed to improve the art of ionization forspectrometry.

Chemical glow discharge ionization methods may utilize variousimplementations of apparatuses to achieve ionization for spectrometryanalysis. Referring to FIGS. 1-3, schematic views of chemical glowdischarge ionization apparatuses 10, 20, 30 are presented. Chemicalionization induced by glow discharge, including the steps of ionizing areagent by glow discharge followed by a charge or proton transfer fromthose reagent ions to vapors of analyte, is utilized by the apparatuses10, 20, 30 of FIGS. 1-3.

Referring specifically to FIG. 1, the chemical glow discharge ionizationapparatus 10 employs glow discharge between electrodes 11 and 12 atabout 1 mBar gas pressure of noble gases—such as Argon or Helium. Thedischarge forms ions and metastable particles Ar⁺ and Ar^(*), which aresampled into reaction chamber 13. Reagent vapors, typical for the CImethod, are introduced into the reaction chamber 13 through line 15 at amuch larger concentration than the analyte vapors, which are introducedinto the reaction chamber 13 through line 14. Reagent ions are primarilyformed by Penning ionization (Ar^(*)+R→Ar+R⁺). In turn, reagent ionstransfer charge to analyte vapors and produce analyte ions, typicallyM−H⁺ and their fragments Fr⁺ (R⁺→M−H⁺, Fr⁺) with spectra very similar toCI for analysis by mass spectrometer 18. The method, which is summarizedin icon 19, has been developed to avoid degradation of electron emittersin C. I. Hunt, et al., Positive and Negative Chemical Ionization MassSpectrometry Using a Townsend Discharge Ion Source, ANAL. CHEM., vol.47, pg. 1730 (September 1975), incorporated fully herein by reference,provides additional information regarding a similar method to that whichis carried out by apparatus 10.

Referring specifically to FIG. 2, the chemical glow discharge ionizationapparatus 20 induces glow discharge in the chamber 22, aided byelectrode 21, to produce large currents of ionizing particles. Watervapors are intentionally introduced, through reagent line 25, into adrift chamber 23, thus promoting formation of water clusters andarranging proton transfer reaction (PTR) to analyte vapors in reactorchamber 27. The reactor 27 employs a medium-strength electrostatic fieldE pressurized to an mBar pressure range to improve ion transfer withinan elongated reactor for breaking larger water clusters W_(n)H⁺ to WH⁺.Thus, the reactor 27 reduces proton affinity of the reagent ion, andthis way enhances ionization of analytes, which are introduced into thereactor chamber 27 through analyte line 24, with moderate protonaffinity in the processes WH⁺→MH⁺. The ionized analytes may be analyzedin mass spectrometer 28. The method, which is summarized in icon 29, hasbeen developed for detection of ultra-traces in air and for breathanalysis. Hansel, et al., Proton Transfer Reaction Mass Spectrometry,INT'L J. MASS SPECTROM. & ION PROC., vol. 149, pg. 609 (1995), which isfully incorporated herein by reference, provides additional informationregarding a similar method to that which is carried out by apparatus 20.

Referring specifically to FIG. 3, the chemical glow discharge ionizationapparatus 30 employs liquid nebulization and evaporation within chamber32, which receives sample S through analyte line 34. The chamber 32samples analyte vapors and solvent into a glow discharge chamber 33containing Helium at atmospheric pressure. Water traces in the gas actas a reagent to form water cluster ions, thus promoting soft ionizationof analyte at RH+→MH+ processes within the chamber 33 and aided byelectrode 31. The ionized sample is introduced into mass spectrometer 38through a differentially-pumped orifice 36. Adding water solvent withliquid samples substantially improves softness for organic spectra.Lubman, et al., Liquid Sample Injection Using an Atmospheric PressureDirect Current Glow Discharge Ionization Source, ANAL. CHEM., vol. 64,pg. 1426 (1992), which is fully incorporated herein by reference,provides additional information regarding a similar method to the methodthat is carried out by apparatus 30, which is summarized in icon 39.Exposure of analyte vapors to glow discharge also causes some moderatefragmentation. The proton transfer reactions caused non-linear signalper concentration response, resulting in non-uniform ionization.Efficiency of ionization of the method carried out by apparatus 30 canvary within three orders of magnitude between analyzed compounds.

Proton affinity depends on compound polarity, which explains thenon-uniform ionization between chemical classes. Operation atatmospheric, rather than operating at mbar pressures, increases the roleof ion molecular reactions, which explains mutual analyte interferencesand matrix suppression effects, even at large excess of the chargingagent. Thus, methods of chemical ionization induced by glow discharge inapparatuses 10, 20, 30 are relatively soft (though not being as soft asESI or APCI which do not expose analyte to glow discharge), but is nottruly quantitative.

Referring to FIG. 4, a conditioned glow discharge ionization apparatus40 employs a capillary 45 for conditioning glow discharge plasma betweenglow discharge chamber 42 and reactor chamber 43. A fine (e.g., 1.5-2 mmdiameter) and sufficiently long (e.g., 10-20 mm) capillary 45: (a)screen the reactor chamber 43 from the strong electric field, (b) chillthe sampled gas, and (c) remove at least fast electrons to leave ionsand metastable discharge ions that produce analyte ions from sampleinjected through an orthogonal-set sample transfer line 44 and mixedwith conditioned glow discharge products in the reactor chamber 43,which is typically 5-10 mm in diameter and length. The products aresampled into a mass spectrometer 48 via an aperture 46, which is usuallysized with a 0.5-2 mm diameter. WO 2012/024570 by the present inventors,which is fully incorporated herein by reference, provides additionalinformation regarding a similar method to the method that is carried outby apparatus 40, which is summarized in icon 49.

The method of FIG. 4 appears very sensitive to minor (at ppm level)impurities or traces in the auxiliary gas, to minor seal leaks, and toimpurities in the source materials. This also makes the method sensitiveto glow discharge regimes (arc versus glow discharge). As a result,so-called quenching of plasma components by chemical impurities definesboth sensitivity and softness of the method, which makes it inconvenientfor routine use.

In contrast to the ionization methods accomplished by apparatuses 10,20, 30, 40, implementations of the present disclosure are directedtoward providing a soft and quantitative ionization method, which may bereferred to as a Cold Conditioned Glow Discharge method. Turning toFIGS. 5A-5B, schematic views of example conditioned glow dischargeionization mass spectrometry apparatuses 50 are illustrated forimplementing mass spectrometric analysis of complex mixtures ofsemi-volatile compounds utilizing the ionization method of the presentdisclosure. The apparatus 50 includes a coaxial discharge tubularelectrode 51, a glow discharge chamber 52, a reactor chamber 53 housinga sampling conditioning channel 55, a sampling nozzle 56, and a massspectrometer 58 with differential pumping system and an ion transferinterface (not shown in FIGS. 5A-5B). Further, the apparatus 50 includesa quartz capillary 54, which supplies the analyzed sample S. The quartzcapillary 54 protrudes through the discharge electrode 51, through theglow discharge chamber 52, and through the sampling conditioning channel55 and ends in the close vicinity or within the throttle of the samplingnozzle 56. In alternate implementations of the apparatus 50, the tip ofthe capillary 54 may protrude into the nozzle. To form desired gas flowsand a desired gas pressure range of about 3 to 100 mBar in the glowdischarge chamber 52, a technically pure (5.0 or 6.0) noble gas (e.g.,Argon) is fed to the discharge chamber. The gas is evacuated past thesampling nozzle 56 by a mechanical pump 57, which may operate at a pumpspeed of 5 L/s or higher. The mechanical pump may be supplied with anoil filter (not shown in FIGS. 5A-5B).

FIG. 5B illustrates a conditioned glow discharge ionization massspectrometry apparatus 50 b similar to the apparatus 50 a of FIG. 5A.The apparatus 50 b differs from the apparatus 50 a in that the apparatus50 b includes an arrangement of the sample conditioning channel 55 bformed with an inserted tube 60, unlike the tubeless sample conditioningchannel 55 a of the apparatus 50 a. In operation, a noble gas (e.g.,Argon) is fed at a sufficient rate (e.g., between 100-1000 mL/min) tosustain gas pressure between 3 and 100 mbar (e.g., between 10 and 20mBar), and the nozzle aperture is sized between 0.3 mm and 3 mm (e.g.,between 1 mm and 2 mm). An RF or DC voltage in the kilovolt range isconnected to electrode 51 via a ballast resistor in the range from0.3-3.0 MOhm to induce a glow discharge between electrode 51 and agrounded counter-electrode, which is formed either by the walls of theionization chamber 52, by the reactor chamber 53, or by a conditioningtube 60. The quartz capillary 54 with removed polyimide coatingprotrudes through the tubular electrode 51 and sampling conditioningchannel 55. The capillary 54 may stabilize the glow discharge regime dueto surface discharges. The gas flow samples the glow dischargeproducts—including Argon ions Ar⁺, Argon metastables Ar^(*), andelectrons e—through the reactor chamber 53. Fast electrons charge theinsulated quartz capillary, this way inducing a radial electric fieldthat pushes charged particles to walls and leaves metastable atomswithin the flow. According to literature, metastable Ar^(*) atoms livefor about 1 second, while transfer time in the interface is between10-30 ms. The metastable Ar^(*) excitation energy is 13.6 eV which isenough for Penning ionization of the analyte with any smaller ionizationpotential expressed as follows.

Ar^(*)+M→M⁺+e+Ar for PI(M)<13.6 eV   (1)

In some implementations, Argon metastable atoms are coaxially mixed withanalyte molecules in the close vicinity or at the throttle of thesampling nozzle 56. Coaxial mixing preserves a high concentration of theanalyte within the gas flow. At 1 mm-2 mm nozzle size and 5-10 L/spumping speed of the fore-vacuum pump 57, a supersonic jet forms (such ajet forms at the nozzle pressure ratio above 2), which inevitably is acooling gas jet and provides some cooling of the entrained analytemolecules. As in Cold EI methods, vibrational cooling of this typenotably reduces ion fragmentation. Accordingly, gaseous cooling withinthe supersonic jet improves the softness of the described method ofconditioned glow discharge (i.e. a Cold CGD). Cooling is particularlyeffective, since the entire source has to be at 250-280° C. to avoidsample accumulation at surfaces.

The exemplary apparatus and CGD method differ from the CGD methoddisclosed in WO 2012/024570 by at least five features and processes: (i)coaxial supply of analyzed sample to concentrate the sample on the flowaxis; (ii) stabilizing of glow discharge regime by coaxial quartzcapillary protruding through the glow discharge region; (iii) plasmaconditioning within a channel in presence of low conductive or insulated(at 250° C.) quartz capillary which promotes removal of chargedparticles; (iv) minimizing time and number of ion molecular reactionswithin the reactor by introducing quartz capillary close to the nozzlethrottle; and (v) local cooling of analyte molecules at the time of theanalyte ionization. Due to the removal of charged species in theconditioner, Penning ionization by metastable Argon atoms becomes themain ionizing channel. In some implementations, Helium is utilized toproduce long living metastable atoms for providing a similar ionizationmechanism and source analytical parameters with somewhat harsherionization.

The Cold CGD method of the present disclosure includes the followingsteps: (a) arranging an enclosed ionization chamber 52 of technicallyclean materials (stainless steel, ceramics, copper and graphite seals)for minimal outgasing at 250-300 C; (b) heating said ionization chamber52 to at least 250 C; (c) feeding technically pure (at least 5.0 andpreferably 6.0) noble gas into said ionizing chamber 52 at flow ratesbetween 100 to 1000 mL/min; (d) sampling said gas into a nozzle 56followed by a fore-vacuum pump 57 to arrange a gas flow through thetubular counter-electrode 55 and to sustain gas pressures between 5 and100 mBar in said ionizing chamber 52; (e) inducing either RF or DC glowdischarge at 0.3-10 mA current between tubular electrode 51 and acounter-electrode; (f) stabilizing glow discharge regime by inserting aninsulating surface, such as bare quartz capillary 54, through saidtubular discharge electrodes 51; (g) sampling discharge products fromthe discharge region into a counter-electrode by a gas flow through aconductive plasma conditioning channel 55 or 55B with diameter between 1and 3 mm and length between 5 and 30 mm; (h) protruding bare quartzcapillary 54 through said plasma conditioning channel 55 or 55B forremoval of charged particles by the electrostatic field of the chargedinsulating surface; (i) supplying analyzed sample via said quartzcapillary at flow rates in the range from 1 to 100 mL/min; (j) samplingglow discharge products and mixing analyzed sample with a conditionedplasma flow in close vicinity or in the throttle of a sampling aperture56; (k) providing local cooling of the analyte within a supersonic gasjet, formed within and past the aperture 56; (1) sampling reactionproducts into a mass spectrometer 58, preferably via an intermediatepumping stage in presence of confining radio-frequency fields.

In some implementations the exemplary CGD method is applied for massspectrometric analysis of complex mixtures of semi-volatile compounds.Components are time separated within a gas chromatograph, ionized in theCGD source, and analyzed in a mass spectrometer. Formation of M+ ionsonly ease spectra interpretation. The mass spectrometric method may befurther improved by inducing fragmentation of M+ ions by injecting saidions into a radio-frequency ion guide at elevated ion energy. Sincefragmentation patterns and fragment types are primarily defined by ionstructure, fragment spectra appear very similar to fragments in theelectron impact ionization. This allows identifying analyte molecules byboth molecular mass information and by fitting fragment spectra to NISTlibrary.

The CGD method may provide excellent bases for quantitative information.As described in greater detail below, the ionization efficiency isuniform across wide range of compounds. The signal response remainslinear at least within four orders of magnitude, regardless of chemicalmatrix with up to at least 10 ng/sec flux.

Turning to FIG. 15, an exemplary arrangement of operations for a methodfor assembling a mass spectrometry apparatus is illustrated. At block1502, the method includes providing a reactor chamber 53 defining asampling conditioning channel 55. At block 1504, the method includesarranging a glow discharge chamber 52 adjacent to the reactor chamber53. At block 1506, the method includes providing a tubular electrode 51extruding into an interior of the glow discharge chamber 52. At block1508, the method includes arranging a sampling nozzle 56 at an outletend of the reactor chamber 53. At block 1510, the method includesproviding a quartz capillary 54 for sample introduction. The quartzcapillary 54 passes through the glow discharge chamber 52, protrudesthrough the tubular electrode 51, and passes at least partially throughthe reactor chamber 53. At block 1512, the method includes arranging amechanical fluid pump 57 in a location to allow evacuation of gas fromthe glow discharge chamber 52. The evacuation of the gas by themechanical fluid pump 57 passes the gas by the sampling nozzle 56.

Role of Collisional Cooling In Supersonic Jet

Referring to FIGS. 6A and 6B, softness of the exemplary CGD method isassisted by the analyte cooling in the supersonic jet past the nozzle56. The plotted representations of FIGS. 6A and 6B present profilesobtained by moving the tip of the capillary 54 relative to the throttleof the supersonic nozzle 56. Measurements have been done at variousArgon fluxes into the source at a 1 mm-diameter-sized nozzle 56. Theplot of FIG. 6A presents the profile of relative intensity of M+ octaneions versus total Octane spectrum intensity, which reflects theionization softness. The plot of FIG. 6B presents profiles of signalabsolute at 4 ng octane injections. Within the alkane class, small sizemolecules appear more fragile, and it takes more efforts obtainingstrong relative intensity of M+ ion, while higher alkanes are primarilypresented by molecular ions. Looking back at softness profiles, thesoftness improves when capillary reaches the nozzle 56 or protrudes thenozzle 56, and at higher Argon fluxes above 650 mL/min the softnessimproves 3-4 times (i.e. from 2-3% to 10-12%). Apparently, the maximumtotal ionic signal is also observed when capillary is placed right intothe nozzle throttle. Compared to a 20 mm distance from the nozzle, thesignal grows almost 10-fold. The effect may be explained by rapidspace-charge expansion of ions formed in the upstream reactor, whileions formed in the nozzle throttle and in the axis of the jet wouldremain focused and are better transferred within the interface. Thus,the arrangement with coaxial placement of quartz capillary 54 at thenozzle 56 throttle improves both softness and sensitivity of the CGDmethod.

Identification of Analytes with the Exemplary CGD Method

Referring to FIG. 7, for a wide range of semi-volatile compounds, theexemplary CGD method forms molecular M+ ions without forming protonatedMH+ ions, even for classes with large proton affinities, such asphthalates (see icon 730) and nitroses (see icon 750). This results insimplified spectra interpretation (compared to photo-ionization formingM−H⁺, M⁺ and M+H⁺ions) and avoids competition for proton, which is knownto produce discrimination and matrix effects. In the example shown, themost notable effect is low fragment intensity for larger size alkanes(saturated hydrocarbons SHC, here C₂₀H⁴², see icon 740) and forphthalates. The EI spectra for those compounds produces negligibly smallmolecular peaks. Known quantitative and semi-quantitative methods, likePI and Cold EI, do not form such intensive molecular peaks for alkanesand also form mixed types of molecular ions. Thus, the disclosed CGDmethod appears unique in that it forms primarily M+ ions and it is soft(i.e. produces very few fragments).

For the purpose of analyte identification, molecular M+ ions may befragmented within an ion transfer interface or within a collisionaldissociation (CID) cell of a tandem mass spectrometer. Referring to FIG.8, the exemplary plots 810, 820, 830 show that fragmentation has beeninduced by increasing ion energy at the entrance of RF ion guide at 10mTor gas pressure. When ion injection energy has been kept close to zero(plot 810), hexachlorobenzene (HCB C₆Cl₆) spectra were presented bymolecular ions only. When raising energy to 30 eV (plot 820), minimalamount of fragments M−Cl+ appeared; at 40 eV (plot 830) fragments becamemore intense than molecular ion. Thus, at CID fragmentation of M+ ions,fragment ions are similar to EI fragments and spectra could be matchedwith NIST library.

Referring to FIGS. 9-10, optimal fragmentation energy depends on theanalyte structure as shown by representative curves for six aromaticcompounds on FIG. 9, shows a plot of softness (relative intensity ofmolecular M+ ions to the total spectrum intensity) versus ion injectionenergy into RF ion guide. Nevertheless, as shown in FIGS. 10A-10D,standard NIST identification provides high scores around 700-800 over awide range of fragmentation energies. FIG. 10A compares experimentalspectrum of 1,2-dichloro benzene with an NIST spectrum, which isreflected at the bottom part of the plot. The NIST score is 846.Constraining molecular weight and elemental composition (usuallyautomatically derived at high accuracy measurement of molecular weight),the answer appears first in the search list. FIGS. 10B-10D show how thescore and the rank depend on the fragmentation energy, E, for threeother compounds. For instance, the score remains around 800, and thecorrect answer has either rank one, or the first answer defines aspatial (not structural) isomer, as in case of dibenzofurane in FIG.10D. Thus, the disclosed CGD method provides both molecular weight andstructural identification within wide range of fragmentation energiesand for wide variety of analyte compounds.

Application Example

The CGD source is expected to be particularly useful for petroleomicsand metabolomics. Referring to FIG. 11, for an analysis mixture of fattyacids methyl esters (FAME), the CGD method provides a reasonably uniformresponse between compounds of the same class as seen from total ioncurrent (TIC) trace (plot 1110). The CGD spectra of FAME are presentedby M+ ions with about 10% of intensity of all fragments (plot 1120).Fragment ion masses match those in EI (plot 1130).

Quantitative Analysis

FIGS. 12A-12C compares a total ion current for the CGD and EI methodsfor a MegaMix sample (Restek) mixture. Since mixtures may degrade atstorage, both analyses were made with the same sample at the same day.Signals correlate well between those two methods as seen from TICcomparison of FIG. 12A (CGD method on top and EI reflect below) and fromthe plot of FIG. 12B (presenting ration of TIC for the CGD and EImethods). Since the EI method is recognized as the golden standard forquantitative analysis, the above correlation indicates uniformionization efficiency of the exemplary CGD method across wide range ofclasses, including PAH, PCB, phthalates, nitro and oxy compounds, andfor moderate size chlorinated compounds. The correlation presented inFIG. 12C provides additional confirmation. The collector current pastCGD source (shown on top) is compared to mass spectrometric signal TIC(shown below). Both correlate well to assure that there no significantdiscriminations at MS measurements, for example, such as low mass cutoff in the RF transfer interface. Overall, FIG. 12C shows that the CGDsource ionization efficiency (ratio of produced ions per injectedmolecules) can be estimated as IE-3, since 10 ng injections wereproducing 20 nC charge at the collector. Moderate ionization efficiencyis chosen to match the capacity of the transfer interface and thedynamic range of MS detector, while reducing sensitivity to impuritiesin the source materials and gases.

FIG. 13 shows a plot of bi-logarithmic signals versus HCB load onto agas chromatograph (GC) column illustrating capabilities of CGD forquantitative analysis. In the example shown, the CGD mass spectrometricsignal is substantially linear with an amount of injected sample withinfour orders of magnitude with correlation coefficient R=0.99999. At evenhigher loads of 100 ng into the GC column, the signal linearity drops bya factor of two, indicating some saturation processes. Measurements ofion currents past the source have indicated that the saturation at highloads is more likely to occur within the ion transfer interface, e.g.,by space charge effects within an RF quadruple at ion currents above 20nA (see FIG. 12C).

Analytical Summary

Overall, the exemplary CGD method provides a unique combination ofanalytical properties: (a) the method is soft for wide range of testedsemi-volatile compounds, including the compounds which form negligiblemolecular peak in the EI method; (b) the CGD method forms primarilymolecular ions, which ease spectra identification, since there are noother types of quasi-molecular ions; (c) optionally induced CIDfragmentation allows NIST identification with high confidence,particularly when applying constrain on the molecular mass, known frommeasurements without the controllable fragmentation; (d) ionizationefficiency is fairly uniform across wide range of chemical classes,which is very attractive at quantitative analysis, particularly whenstandards are not available; (e) the ionization efficiency staysconstant and the signal remains linearly proportional to concentrationin at least four orders of dynamic range.

The latter two properties provide good bases to absence of matrixdiscrimination and mutual interference effects at sample fluxes under 10ng/sec.

The novel features of the disclosed CGD method allow a novel genericanalytical method to be formulated. The novel generic analytical methodincludes the following steps: (a) quantitative and soft ionization in aconditioned glow discharge ion source; (b) alternated in timemeasurement of molecular mass and fragmentation of molecular ions; and(c) compound identification by comparing with a library of electronimpact spectra.

Range of Volatility

Analyte volatility for the disclosed CGD method is range-limited simplydue to the method's coupling to a gas chromatograph, where mostnon-volatile compounds would not pass without chemical modifications.However, based on the above measurements of the upper load being 10ng/sec, the disclosed CGD method may retain its analytical merits atmoderate load levels of solvents or matrix up to 10 ng/sec. Thus, thedisclosed CGD method is compatible with additional methods of sampleinjection, such as: (a) direct thermal desorption; (b) desorption bylaser; (c) sample nebulization with sampling of aerosol via curtain gas(preventing sampling of the vast majority of the solvent); and (d)liquid spray at liquid fluxes under 10 nL/min from either CE or nano-LC.

Formulation of the CGD Method

While analyzing multiple processes occurring in the CGD source novelfeatures responsible for analytical merits include: (a) conditioning ofthe glow discharge at fore-vacuum gas pressure achieved by sampling glowdischarge products in the gas flow and passing them through a coaxialchannel with insulating capillary on axis, providing effective removalof charged particles, while effectively transferring metastable atoms ofnoble gases; and (b) injecting a sample into a flow of metastable atomsin close vicinity of the throttle of the supersonic nozzle. The processof injecting the sample (b) causes several positive effects: (i)effective focusing of the analyte on the jet axis and ensures aneffective ion transfer in the subsequent transfer interface; avoidscontact of analyte with the source walls; and (ii) provides vibrationalcooling of the analyte, which in turn assists better softness of theionization method.

Turning to FIG. 16, an exemplary arrangement of operations for a methodof mass spectrometric analysis undertaken by the conditioned glowdischarge ionization mass spectrometry apparatus 50 is illustrated. Atblock 1602, the method includes producing either RF or DC glow dischargewithin a noble gas at a gas pressure between 3-100 mBar. The glowdischarge is produced in a glow discharge chamber 52 between a coaxialdischarge tubular electrode 51 and a counter-electrode. The noble gasmay be implemented as a variety of noble gases, such as Helium, Argon,or Krypton. At block 1604, the method includes sampling and conditioningthe glow discharge products within a gas flow while transferring excitedRidberg atoms. The gas flow is driven by a mechanical fluid pump 57. Theglow discharge products are sampled and conditioned through a conductivechannel 55. At block 1606, the method includes mixing the conditioneddischarge products with analyte at elevated temperatures (i.e. above150° C.) to produce a Penning reaction between analyte molecules and theRidberg atoms. This mixing occurs in an enclosed chamber and generatesions of the analyte. At block 1608, the method includes sampling theanalyte ions for mass spectrometric analysis in the mass spectrometer58. A gas flow samples the into the mass spectrometer 58. Generally, themethod may be improved with one of the following additional steps: (a)removing charge within the sampling conditioning channel 55 by chargingof an insulating surface protruding through the sampling conditioningchannel 55; (b) coaxially mixing the analyte flow with the flow ofconditioned plasma; or (c) cooling the mixed flow within a sonic orsupersonic jet for reducing the region of Penning ionization to coldjet.

Enhanced Peak Capacity

As a further extension of analytical utility, the CGD source has veryrapid reaction time. The flow is arranged without pockets and the sourcewalls avoid lengthy analyte absorption. Yet, there is no additional peaktailing, and the source is compatible to fast GC×GC separation, similarto the glow discharge source of WO 2012/024570 (CGD), which is fullyincorporated herein by reference. The combined power of (a) GC×GCseparation with typical 10,000 peaks capacity and (b) soft andquantitative ionization in the CGD source, would particularly shine fora high resolution multi-reflecting TOF mass spectrometer (MRTOF−MS).Preferably, the high resolution MRTOF−MS employs a collisional dampeninginterface and a double orthogonal accelerator, operating in the regimeof frequent encoded pulsing (EFP) as described in WO 2005/001878(MRTOF), WO 2007/044696 (OA), and WO 2011/135477 (EFP), each of which isfully incorporated herein by the reference. Such combination is expectedto have an overall peak capacity over 10 million, though not accountingfor redundancy between chromatographic and accurate mass separationtechniques.

As already described, the CGD source is at least compatible with GC andCE upfront separations, both by the speed and by the flux of matrix andcarrier fluids. Thus, the advantage of high peak capacity is expectedalso in cases of using such chromatographic tandems as: (i) GC×GC; (ii)LC−GC; (iii) LC−LC; (iv) LC−CE.

Enhanced Selectivity

As another extension of analytical utility, selectively is enhanced whenutilizing the disclosed CGD method. Simply, users want to see targetcompounds at maximal selectivity, reliability, and sensitivity, whilethey do not want additional information on the rich matrix andaccompanying analyte components. Target analysis with CGD ion sourcecould be enhanced in multiple ways. First, selectivity of ionizing someparticular analyte classes may be enhanced by adding a reagent gas intothe volume of conditioned plasma. In one example, acetone reagent wouldpromote the soft ionization of polar molecules. Additional small flowmay be added, for example, into the carrier Helium gas of gaschromatograph or into the source reagent zone via separate line. Second,molecular ions could be mass selected by either a mass filter (such asquadrupole mass filter) or in a mobility separator. Third, theselectivity and separation capacity of the method could be enhanced byan ion mobility separation. A complimentary analytical power occurs whensuch separation methods are combined with quantitative and softionization. Finally, both separation power and analysis selectivity ofCGD mass spectrometric analysis may be enhanced if alternating orscanning the fragmentation energy.

1. A method of mass spectrometric analysis, the method comprising thefollowing steps: producing either RF or DC glow discharge within a noblegas at gas pressure between 3 and 100 mBar; sampling and conditioningglow discharge products within a gas flow through a conductive channelfor removing charged particles while transferring excited Ridberg atoms;mixing conditioned discharge products with analyte flow within anenclosed chamber at elevated temperatures above 150° Celsius forproducing Penning reaction between analyte molecules and said Ridbergatoms to generate ions of said analyte; sampling by a gas flow saidanalyte ions for mass spectrometric analysis; and at least one of thefollowing steps: (i) removing charge within said conditioning channelcaused by charging of an insulating surface protruding through saidconditioning channel; (ii) coaxially mixing of analyte flow with theflow of conditioned plasma; and (iii) cooling of said mixed flow withina sonic or supersonic jet for reducing the region of Penning ionizationto cold jet.
 2. The method of claim 1, wherein said molecular ions ofanalyte are partially fragmented either at an ionization event or at astep of controllable collisional induced dissociation, wherein obtainedfragment spectra are compared with library spectra of electron impactfor analyte identification and for a structure elucidation.
 3. Themethod of claim 2, further comprising introducing less volatilecompounds within a liquid or solid matrix to extend the range of analytevolatility whereat a matrix flux remains under 10 ng/sec and where theanalyte sample is brought to a gas phase bone one of the followingsteps: (a) applying a rapid thermo desorption; (b) applying pulsed laserdesorption; (c) applying nebulization a liquid sample with removal ofsolvent vapors by side or counter gas while passing through aerosol; and(d) applying nebulization at a flow rate under 10 nL/min pas capillaryelectrophoresis or nano liquid chromatography.
 4. A method as in claim1, further comprising a step of upstream tandem chromatographicseparation of the list comprising: (i) GC×GC; (ii) LC−GC; (iii) LC−LC;(iv) LC−CE.
 5. A method as in claim 2, for the purpose of enhancedselectivity at analysis of complex mixtures, further comprising a stepof mass or ion mobility selection of parent ions prior to the step ofion fragmentation.
 6. A method as in claim 2, further comprising a stepof varying fragmentation energy.
 7. A method as in claim 1, for thepurpose of enhanced analysis selectivity further comprising a step ofadding a reagent gas into the region of conditioned glow discharge, thusconverting Ridberg ions into reagent ions.
 8. A method as in claim 1,further comprising a step of mass-spectrometric analysis of ionizedanalyte ions with a high resolution multi-reflecting time-of-flight massspectrometer, operating in the regime of frequent encoded pulsing.
 9. Amethod of mass spectrometric analysis comprising the following steps:quantative and soft ionizing in a conditioned glow discharge ion source;alternated in time measuring of molecular mass and fragmentation ofmolecular ions; and compound identifying by comparing with library ofelectron impact spectra.
 10. A method as in claim 9, further comprisingat least one step of the group: (i) an upfront multi-stagechromatographic separation of analyte molecules; (ii) an upfront massseparation of molecular ions; (iii) ion mobility separation of molecularions.
 11. A method for assembling an ion source for a mass spectrometryapparatus, comprising: providing a reactor chamber defining a samplingconditioning channel; arranging a glow discharge chamber adjacent to thereactor chamber; providing a tubular electrode extruding into aninterior of the glow discharge chamber; arranging a sampling nozzle atan outlet end of the reactor chamber; providing a capillary for sampleintroduction, wherein the capillary passes through the glow dischargechamber, protrudes through the tubular electrode, and at least partiallypasses through the reactor chamber; and arranging a mechanical fluidpump in a location to allow evacuation of gas, wherein the mechanicalfluid pump evacuates the gas from the glow discharge chamber past thesampling nozzle.
 12. The method of claim 11, further comprising:charging an insulated surface that protrudes through the samplingconditioning channel.
 13. The method of claim 11, wherein the gasevacuated by the mechanical fluid pump comprises a noble gas, andwherein the noble gas is pressurized within the glow discharge chamberprior to evacuation.
 14. An ion source for a mass spectrometryapparatus, comprising: reactor chamber defining a sampling conditioningchannel; a glow discharge chamber residing adjacent to the reactorchamber; a tubular electrode extruding into an interior of the glowdischarge chamber, wherein the tubular electrode receives a voltage; asample nozzle residing at an outlet end of the reactor chamber; acapillary for sample introduction, wherein the capillary passes throughthe glow discharge chamber, protrudes through the tubular electrode, andat least partially passes through the reactor chamber; and a mechanicalfluid pump residing in a location to allow evacuation of gas, whereinthe mechanical fluid pump evacuates the gas from the glow dischargechamber past the sampling nozzle.
 15. The ion source of claim 14,further comprising: an insulated surface that protrudes through thesampling conditioning channel.
 16. The ion source of claim 14, whereinthe gas evacuated by the mechanical fluid pump comprises a noble gas,and wherein the noble gas is pressurized within the glow dischargechamber prior to evacuation.
 17. An analytical method, comprising:quantitatively soft ionizing an analyte in a conditioned glow dischargeion source; alternating-in-time measuring of molecular mass andfragmentation of molecular ions; and identifying compounds by comparingwith a library of electron impact spectra.
 18. The analytical method ofclaim 17, wherein the ionizing step comprises: producing a glowdischarge from a noble gas at an elevated pressure between 3-100 mBar;conditioning the glow discharge through a sampling conditioning channel;and ionizing an analyte by mixing the analyte with the conditioned glowdischarge at an elevated temperature.
 19. The analytical method of claim18, wherein an insulated surface within the sampling conditioningchannel is charged to remove charge of a flow through the samplingconditioning channel.
 20. The analytical method of claim 18, wherein thestep of ionizing an analyte comprises a coaxial mixing of a flow of theanalyte with a flow of the glow discharge.
 21. The analytical method ofclaim 18, wherein a step of cooling the ionized analyte within a jetoccurs after the step of ionizing an analyte.
 22. The analytical methodof claim 18, wherein the step of ionizing an analyte occurs at thethrottle of a sampling nozzle.
 23. A method of conditioned glowdischarge ionization, comprising: arranging an enclosed ionizationchamber for minimal outgassing; heating the ionization chamber to anelevated temperature above 250 degrees Celsius; feeding a noble gas intothe ionization chamber at a flow rate between one hundred millilitersper minute and one thousand milliliters per minute; evacuating the gasthrough a sampling conditioning channel; inducing glow discharge betweena tubular electrode and a counter-electrode; inserting an insulatingsurface through the tubular electrode to stabilize the glow dischargeinducement; creating a gas flow through the sampling conditioningchannel to move the induced glow discharge products; protruding theinsulating surface through the sampling conditioning channel;introducing an analyte sample; mixing the analyte sample with the glowdischarge products in close vicinity or within a throttle of a samplingaperture; providing local cooling of the analyte sample within a gascooling jet, wherein the gas cooling jet is formed within or around thesampling aperture; and directing products to a mass spectrometer. 24.The method of claim 23, wherein the insulating surface comprises aquartz capillary, and wherein the quartz capillary introduces theanalyzed sample.